Intercalation of anticancer drug Palbociclib with calf-thymus DNA: new insights from molecular spectroscopic, molecular dynamic simulations and cleavage studies.

The interaction between the anti-cancer drug Palbociclib (PAL) and calf-thymus DNA (CT-DNA) was investigated using various biophysical techniques in a physiological buffer (pH 7.4). It was found that PAL intercalated into the base pairs of CT-DNA as evidenced from the results of UV-Vis, fluorescence, circular dichroism (CD), competitive binding assay with ethidium bromide (EB) and Hoechst 33258, KI quenching study, the effect of denaturing agent and viscosity measurements. The magnitude of binding constants (106 M-1) at different temperatures suggested strong binding between PAL and CT-DNA during complexation. The observed ΔHo > 0 and ΔSo > 0 indicated that the binding process is primarily driven by hydrophobic interactions. Molecular docking studies indicated partial intercalation of pyridopyrimidine ring between the base pairs of DNA. Free energy surface (FES) analysis derived from metadynamics simulation studies revealed the PAL-induced cleavage of DNA, which was confirmed by gel electrophoresis experiments.Communicated by Ramaswamy H. Sarma.

Spectroscopic and Theoretical Studies on the Selective Detection of Cyanide Ions by a Turn-On Fluorescent Chemo-Dosimeter and its Application in Living Cell Imaging.

A new chemo-dosimeter AK4 containing quinoline fluorophore has rationally been designed, synthesised and characterized using 1H and 13C NMR and mass spectral techniques. The probe senses explicitly CN- ion through a dramatic enhancement in fluorescence over other commonly coexistent anions in H2O:DMSO (9:1 v/v) medium over a broad pH range (4-10). 1H NMR titration revealed the deprotonation followed by nucleophilic addition reaction of CN-, which was supported by 13C NMR and mass spectral examinations. The Job's continuous variation method indicated the formation of a 1:1 adduct between AK4 and CN- with a binding constant of 1.62 × 104 M-1. A limit of detection (LOD) towards CN- of 0.69 µM has been determined, which is much lower than the World Health Organization (WHO) recommended limit of CN- in drinking water (1.9 µM). The changes in the optical properties of AK4 upon reaction with CN- were delineated using Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations. Moreover, fluorescence microscopic studies established that AK4 could be an effective probe for imaging intracellular CN- in HeLa cells.

Multi-Spectroscopic and TD-DFT Studies on Chromogenic and Fluorogenic Detection of Cyanide in an Aqueous Solution.

Different spectroscopic techniques and Density Functional Theory (DFT)/Time-Dependent Density Functional Theory (TDDFT) calculations have been employed to investigate the dual channel CN- detection behaviour of the developed chemo-dosimeter (AK3). The CN- with AK3 reaction triggered a colour change from pale yellow to colourless and enhanced fluorescence. UV-Vis, fluorescence, 1H & 13C NMR and mass techniques coupled with theoretical calculations (Mulliken charges, dihedral angles) revealed that the CN- sensing process mechanism involves deprotonation of the N-H group followed by nucleophilic addition reaction. Detailed TD-DFT calculations showed that the relaxation of excited electrons from LUMO and to two different ground states is responsible for the weak/moderate fluorescence of AK3. Nucleophilic addition of CN- to the C-atom of the CH = CH bridge terminated the π-conjugation between donor and acceptor regions, reduced the coplanarity, decreased the ICT transition and consequently enhanced the fluorescence of the probe. The practical utility of the probe was demonstrated by detecting cyanide in food materials and determining CN- in environmental water samples.

Multi-spectroscopic, calorimetric and molecular dynamics evaluation on non-classical intercalation of antiviral drug Molnupiravir with DNA.

The interaction of an antiviral drug Molnupiravir (MOL) with calf thymus DNA (CT-DNA) was investigated using a series of biophysical techniques. A significant hyperchromism with a blue shift nm in the UV-Vis spectra indicated a high binding affinity of MOL for CT-DNA with binding constants in the order of 105 M-1. Competitive fluorescent dye displacement assays with ethidium bromide (EB) and Hoechst 33258 suggested an intercalative mode of binding of MOL with CT-DNA. Thermodynamic profiles determined using fluorescence titration and isothermal titration calorimetric (ITC) analysis matched well with each other. The negative free energy change revealed that the MOL/CT-DNA complexation is a spontaneous process. The negative values of enthalpy and entropy changes indicated that H-bonding and van der Walls interactions play dominant roles in stabilizing the complex. A decrease in viscosity of CT-DNA solution upon adding MOL indicated a partial intercalation mode of binding which was well supported by circular dichroism (CD) spectral and effect of KI and denaturation studies. Molecular docking and metadynamics simulation studies clearly showed the partial intercalation of the pyrimidine ring of MOL into the base pairs of DNA. Free energy surface (FES) contour indicated that the drug/DNA complex is stabilized by H-bonding and pi-pi/pi-cation interactions.Communicated by Ramaswamy H. Sarma.

Insilico molecular modelling to identify PDK-1 targeting agents based on its protein-protein docking interaction.

PDK1, an attractive cancer target that downstreams 23 other kinases towards cell growth, survival and metabolism has gaining attention due to allosteric effect of ligands bound to it. Generally, the drug design strategy using pharmacophores is either a single protein structure or ensemble or ligand-based. Apart from these methods, yet another new approach of protein-protein docking with state of art computational tool like Schrodinger Suite to generate pharmacophores based on the interacting partners of the protein is proposed in this work. The structure-based pharmacophoric features were picked up from docking the ten interacting partners of PDK1 and screened against the Enamine libraries containing protein-protein interacting compound collection, advanced, protein mimetic and allosteric compounds. High throughput virtual screening against the PIF pocket of PDK1 yields an indole scaffold. The identified indole derivative is proposed to be a strong activator that binds in the protein-protein interaction site of PDK1 which was further confirmed by molecular metadynamics simulations, free energy surface analysis and MM-GBSA calculations. Thus, the pharmacophores generated by the interacting proteins for PPI can facilitate the virtual screening in structure-based drug discovery of similar therapeutic targets.Communicated by Ramaswamy H. Sarma.

Multi-spectroscopic, thermodynamic and molecular simulation studies on binding of pyrroloquinoline quinone with DNA: coexistence of intercalation and groove binding modes.

The interaction between enzyme-like pyrroloquinoline quinone (PQQ) and calf-thymus DNA (CT-DNA) has been investigated by means of multi-spectroscopic (UV-Vis, fluorescence and circular dichroism), isothermal titration calorimetric (ITC), viscometry and molecular docking and metadynamics simulation techniques. Absorption spectral data suggested the formation of a PQQ/CT-DNA complex, which quenched the fluorescence of PQQ via the dynamic quenching process. The results of CD spectral studies coupled with viscosity measurements, competitive binding assays with Hoechst 33258 and ethidium bromide (EB), KI quenching experiments, gel electrophoresis and DNA melting studies indicated groove binding mode of interaction of PQQ with CT-DNA. ITC experiment revealed that the complex formation is a spontaneous process (ΔGo < 0) with a binding constant of 1.05 × 104 M-1. The observed ΔHo < 0 and ΔSo < 0 pointed out that the complex is stabilized by van der Waals forces along with H-bonding interactions. The outcomes of molecular docking and simulation studies confirmed the binding of PQQ with DNA. The free energy surface (FES) analysis pointed out the existence of an equilibrium between partial intercalation and groove binding modes, which is in good agreement with the competitive binding assays.Communicated by Ramaswamy H. Sarma.

Direct Adsorption of Graphene Oxide on a Glassy Carbon Electrode: An Investigation of Its Adsorption and Electrochemical Activity.

Different modes of attachment of graphene oxide (GO) on an electrode surface resulted in unusual catalytic behavior respective of attachment because of film thickness. The present work investigates the direct adsorption of GO to the surface of a glassy carbon (GC) electrode. Scanning electron microscopy images revealed that multilayers of GO get adsorbed on the GC substrate and the adsorption was limited by folding up of the GO sheets at their edges. π-π and hydrogen bonding interactions between the GO and GC substrate flagged the adsorption of GO. pH studies revealed that higher adsorption of GO was achieved at pH = 3 rather than at pH = 7 and 10. Even though the electroactive surface area of adsorbed GO (GOads) was not remarkable (0.069 cm2), upon electrochemical reduction of GOads (Er-GOads), the electroactive surface area was escalated to be 0.174 cm2. Similarly, the RCT of Er-GOads was boosted to 2.9 kΩ compared to GOads which is 19 kΩ. Open circuit voltage was recorded to study the adsorption of GO on the GC electrode. Multilayered GO best fitted with the Freundlich adsorption isotherm, and the Freundlich constants like n and KF were found to be 4 and 0.992, respectively. The Freundlich constant "n" revealed the adsorption of GO on the GC substrate to be a physisorption process. Furthermore, the electrocatalytic performance of Er-GOads was demonstrated by taking uric acid as a probe. The modified electrode showed excellent stability toward the determination of uric acid.

Multi-spectroscopic and molecular simulation methods of analysis to explore the mode of binding of Mebendazole drug with calf-thymus DNA.

UV-vis, fluorescence, circular dichroism (CD) and 1H NMR spectroscopic techniques have been employed to explore the mode of binding of Mebendazole (MBZ) drug with calf thymus DNA (CT-DNA). UV-vis and fluorescence spectral studies suggested a complex formation between the drug and nucleic acid. The fluorescence of MBZ was found to enhance upon binding with CT-DNA through a ground state complex formation with Kb in the order of 104 M-1. The thermodynamic aspects indicated that the complex formation is a spontaneous process and an entropy-driven one. ΔH0 > 0 and ΔS0 > 0 revealed that hydrophobic interaction plays a dominant role in the stabilization of the complex. Competitive dye displacement assays with ethidium bromide (EB) and Hoechst 33258 dyes and viscosity measurements pointed out that MBZ binds with CT-DNA via intercalation mode, which is confirmed by CD and 1H NMR spectral studies as well as denaturation studies. Molecular docking analysis could not match well with the experimental results. However, molecular simulation studies and the resultant free energy surface (FES) analysis clearly showed that the benzimidazole ring of MBZ intercalated between the base pairs of the nucleic acid, which is in excellent agreement with the results of the various biophysical experiments.

Spectroscopic and TD-DFT studies on the chromo-fluorogenic detection of cyanide ions in organic and aquo-organic media.

A new naked-eye chromogenic and fluorogenic probe KS5 has been developed for the detection of CN- ions in neat DMSO and H2O:DMSO (1:1 v/v) media. The probe KS5 exhibited selectivity towards CN- and F- ions in organic and high selectivity towards CN- ions in aquo-organic media resulting in a colour change from brown to colourless and a turn-on fluorescence response. The probe could able to detect CN- ions via a deprotonation process, which was conceived by consecutive addition of hydroxide and hydrogen ions and confirmed using 1H NMR studies. The limit of detection (LOD) of KS5 towards CN- ions were in the range of 0.07-0.62 µM in both these solvent systems. Suppression of intra-molecular charge transfer (ICT) transition and photoinduced electron transfer (PET) process of KS5 by the added CN- ions are responsible for the chromogenic and fluorogenic changes observed, respectively. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) calculations strongly supported the proposed mechanism along with the optical properties of the probe before and after the addition of CN- ions. To prove the practical applicability, KS5 was successfully utilized to detect CN- ions in cassava powder and bitter almonds as well as to determine CN- ions in various real water samples.

A highly selective probe for fluorometric sensing of cyanide in an aqueous solution and its application in quantitative determination and living cell imaging.

A simple fluorescent probe (KS4) containing multiple reaction sites (phenolic -OH, imine and C = C bonds) is successfully synthesized and characterized using 1H NMR, 13C NMR, mass and single crystal XRD techniques. KS4 exhibits high selectivity towards CN- over a wide range of common anions in H2O:DMSO (1:1 v/v) leading to an amazing turn-on fluorescence at 505 nm via deprotonation of the phenolic -OH group. The limit of detection (1.3 µM) for CN- was much below the standard (1.9 µM) set by the World Health Organization (WHO). Stoichiometry of the interaction between KS4 and CN- was ascertained as 1:1 by the Job's plot method and the binding constant was determined to be 1.5x104 M-1. Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT) based theoretical insight has been appealed to understand the optical properties of KS4 before and after the addition of CN- ion. The probe shows respectable real-time applicability for qualitative detection of CN- in almond and cassava powder as well as quantification in real water samples with excellent recoveries (98.8 - 99.8%). In addition, KS4 is found to safe towards living HeLa cells and successfully applied to the detection of endogenous cyanide ions in HeLa cells.

Investigating binding of insecticide buprofezin to DNA by experimental and metadynamics simulation studies.

Buprofezin (BUP) is an insecticide which belongs to the thiadiazine structural family and known to damage DNA in mice. Though its toxic effect on human is not known clearly, understanding the mechanism of interaction of BUP with DNA can prove useful when required. Multi-spectroscopic experiments such as UV-Vis, fluorescence, circular dichroism (CD) and 1H NMR coupled with viscosity measurements, urea effect and voltametric studies were performed to ascertain the mode of binding of BUP with calf thymus DNA (CT-DNA). Analysis of UV-Vis and fluorescence spectra indicated the formation of a complex between BUP and CT-DNA. Other experiments such as competitive binding assays with ethidium bromide (EB) and Hoechst 33258, viscosity measurements, effect of urea, CD, voltammetric studies and 1H NMR spectral analysis suggested that BUP intercalates into the base pairs of CT-DNA. All these results revealed that the binding mode of BUP with CT-DNA should be intercalation and the binding constant is in the order of 104 M-1. The ΔHo < 0 and ΔSo < 0 suggested that H-bonding or van der Waals force was the main binding force between BUP and CT-DNA. The proposed mode of binding of BUP with CT-DNA has been visualized using in silico molecular docking and metadynamics simulation studies, which showed that the phenyl ring of BUP binds to CT-DNA via π-π stacking interaction in addition to H-bond formation.Communicated by Ramaswamy H. Sarma.

A closer look at the mode of binding of drug pemetrexed with CT-DNA.

The interaction of antifolate drug Pemetrexed (PEM) with CT-DNA has been studied by UV-Vis, fluorescence and circular dichroism spectroscopic techniques. The results of these spectroscopic studies in combination with viscosity measurements, voltammetric and KI quenching studies suggested a less-common mode of binding of PEM with CT-DNA i.e. neither intercalation nor groove binding. Thus, metadynamic (MD) simulation is utilized to decipher the nature of binding of PEM with CT-DNA. Analysis of free energy surfaces obtained in MD simulation, reveals that PEM binds to the 3'- and 5'-ends of the DNA molecule. The thermodynamics of the interaction has been investigated by isothermal titration calorimetric experiment. The analysis shows that PEM binds with CT-DNA strongly with a binding constant of 2.6x109 M-1 and the process is found to be spontaneous (ΔG - 12.84 kcal/mol). Further, positive values of enthalpy (ΔH 6.09 cal/mol) and entropy (ΔS 43.1 cal/mol) changes indicate that the binding is an enthalpically unfavourable and, instead, entropically driven process.Communicated by Ramaswamy H. Sarma.

Intercalation of diafenthiuron insecticide with calf thymus DNA: spectroscopic and molecular dynamics analysis.

A series of biophysical experiments like UV-Vis, fluorescence, circular dichroism (CD), competitive displacement assays, voltammetric studies, viscosity measurements and denaturation effect and metadynamics simulation studies were performed to establish the mode of binding of diafenthiuron (DF) insecticide with calf thymus DNA (CT-DNA). Analysis of absorption and fluorescence spectra in Tris-HCl buffer of pH 7.4 indicates the formation of a complex between DF and CT-DNA and the binding constant of which is in the order of 104 M-1. Competitive displacement assay with ethidium bromide (EB) and Hoechst 33258 suggests that the most probable mode of binding of DF with CT-DNA may be via intercalation mode. The results of other experiments such as CD spectral studies, viscosity measurements and the effect of denaturation agent urea support the intercalation of DF with CT-DNA. Thermodynamic parameters (ΔHo, ΔSo and ΔGo) reveal that hydrogen bonds (H-bonds) or van der Waals (vdW) force is the main binding force in the spontaneous interaction between DF and CT-DNA. Molecular dynamics (MD) simulation studies confirmed the intercalation of DF into the base pairs of CT-DNA.Communicated by Ramaswamy H. Sarma.

Molecular spectroscopic and molecular simulation studies on the interaction of oral contraceptive drug Ormeloxifene with CT-DNA.

The interaction between oral contraceptive drug Ormeloxifene (ORM) and calf thymus DNA (CT-DNA) was studied using UV-Vis, fluorescence, circular dichroism (CD) and 1H NMR spectral techniques under physiological buffer (pH 7.4). Competitive binding assays with ethidium bromide (EB) and Hoechst 33258, viscosity measurements, KI quenching studies, molecular docking and metadynamics simulation studies were also substantiated the spectroscopic results. ORM is found to binds in the minor groove of CT-DNA as evidenced by: (1) non-displacement of EB from EB/CT-DNA complex; (2) appreciable displacement of Hoechst 33258 from its CT-DNA complex; (3) slight alteration in the CD signal; (4) small shifts (Δδ < 0.033 ppm) without broadening in 1H NMR signals and (5) the nearly equal extent of quenching of fluorescence of ORM by KI in the absence and presence of CT-DNA. Negative values of both enthalpy and entropy changes pointed out that the interaction between ORM and CT-DNA is governed mainly by H-bonding and van der Waals forces. Negative free energy change suggested a spontaneous interaction between ORM and CT-DNA. The free energy landscape of the binding process was computed using metadynamics simulation. The simulation study results disclosed that ORM binds to the minor groove of DNA through H-bonding and π-π stacking interactions. The results of molecular docking and simulation studies corroborate the available experimental data.

A highly selective and sensitive ratiometric fluorescent probe for quantitative detection of Al(III) in different natural matrices.

A highly selective and sensitive assay of Al(III) using ratiometric fluorescence enhancement is reported in an aqueous solution. The probe (named RS5) exhibits a red-shift of 54 nm upon binding with Al(III) ion. The significant enhancement response of RS5 at 481 nm is attributed to the formation of a 1:1 complex between the probe and Al(III), wherein RS5 acts as a tridentate NNN-donor ligand. The complexation process is ascertained by1H,13C, and27Al NMR and HR-MS spectral techniques. The binding constant of the complex is determined to be 1.3 × 105M-1. The ratiometric change in fluorescence upon complexation with Al(III) is ascribed to an increase in intramolecular charge transfer (ICT) transition along with chelation enhanced fluorescence (CHEF) processes. The probe can be applied for monitoring Al(III) in a pH range of 6-8. The limit of detection (LOD) of RS5 for the examination of Al(III) is found to be 0.3µM. With an aim to understand the sensing behavior of RS5, the optical properties of the probe and its Al(III) complex are investigated using density functional theory (DFT) and time-dependent density functional theory (TD-DFT) methods. The probe is successfully employed for the determination of Al(III), with very high recovery percentages, in natural matrices like deep well water, tap water, drinking water, pond water, river water, bovine serum albumin (BSA) solution and blood serum.

Multi-spectroscopic and free energy landscape analysis on the binding of antiviral drug remdesivir with calf thymus DNA.

Remdesivir (REM) is an antiviral drug, which exercises its effect by targeting specifically RNA-dependent RNA polymerase. The interaction of REM with calf thymus DNA (CT-DNA) was investigated by multi-spectroscopic techniques (UV-Vis, fluorescence, circular dichroism and 31P NMR) in combination with different biophysical experiments and metadynamics simulation studies. UV-Vis and fluorescence spectroscopic analysis indicated formation of a complex between REM and CT-DNA, whose binding constant is in the order of 104 M-1. Competitive displacement assays with ethidium bromide (EB) and Hoechst 33258 shown that REM binds to CT-DNA via intercalation mode. Significant alteration in the band due to base stacking pairs at 274 nm in the circular dichroism spectrum, appreciable increase in relative viscosity of the biomolecule upon binding with REM and the results of potassium iodide quenching studies confirmed that REM intercalates into the base pairs of CT-DNA. Thermodynamic parameters revealed that the binding of REM to CT-DNA is a spontaneous process (ΔG0 < 0) and the main force which holds them together in the REM/CT-DNA complex is electrostatic interaction (ΔH0 < 0 and ΔS0 > 0). The up-field shift in the 31P NMR signal of REM on interaction with CT-DNA suggested that phenyl ring adjacent to the phosphate moiety of REM may involve in the intercalation process. This is well supported by the analysis of free energy surface landscape derived from metadynamics simulation studies.

Spectroscopic and theoretical evidences for the surface binding of voglibose drug with DNA.

Binding of voglibose (VOG), an alpha glucosidase inhibitor, with CT-DNA has been investigated using various spectroscopic techniques including UV-Vis, fluorescence and circular dichroism (CD) coupled with relative viscosity. Isothermal titration calorimetric studies have been used to calculate the thermodynamic parameters such as ΔH (0.0188 cal/mol), ΔS (63.3 cal/mol/K) and ΔG (-18.8 kcal/mol), which reveal that the binding is a spontaneous process and hydrophobic and H-bonding interactions play major roles in the binding process. Effect of ionic strength confirms the existence of hydrophobic interaction between VOG and CT-DNA. Competitive displacement assays with ethidium bromide (EB) and Hoechst 33258 suggest that VOG possibly binds on the surface of CT-DNA. Viscosity measurements also disclose that the binding could be mainly surface binding. Corroborating the experimental observations, metadynamics molecular simulation studies confirm that VOG binding on the surface of the DNA molecule through hydrophobic interactions and direct and water molecule mediated H-bonding.

Concentration-dependent mode of binding of drug oxatomide with DNA: multi-spectroscopic, voltammetric and metadynamics simulation analysis.

The interaction between antihistaminic drug oxatomide (OXT) and calf-thymus DNA (CT-DNA) has been investigated in a physiological buffer (pH 7.4) using UV-Vis, fluorescence, 1H NMR and circular dichroism spectral techniques coupled with viscosity measurements, KI quenching, voltammetry and in silico molecular modeling studies. OXT binds with CT-DNA in a concentration-dependent manner. At a lower [Drug]/[CT-DNA] molar ratio (0.6-0.1), OXT intercalates into the base pairs of CT-DNA, while at a higher [Drug]/[CT-DNA] molar ratio (13-6), the drug binds in the minor grooves of CT-DNA. The binding constants for the interaction are found to be in the order of 103-105 M-1, and the groove binding mode of interaction exhibits a slightly higher binding constant than that of intercalative mode. Thermodynamic analysis of binding constants at three different temperatures suggests that both these modes of binding are mainly driven by hydrophobic interactions (ΔHo > 0 and ΔSo > 0). Voltammetric investigations indicate that the electro-reduction of OXT is an adsorption controlled process and shifts in reduction peak potentials reiterate the concentration-dependent mode of binding of the drug with CT-DNA. The free energy landscape obtained at the all-atom level, using metadynamics simulation studies, revealed two major binding forces: partial intercalation and minor groove binding, which corroborate well with the experimental results.Communicated by Ramaswamy H. Sarma.

Spectroscopic Studies on the Interaction of Naphthyridines with DNA and Fluorescent Detection of DNA in Agarose Gel.

Four new naphthyridine derivatives (R1-R4) possessing amino acid or boronic acid moieties have been synthesized and characterized using 1H and 13C NMR, FT-IR, and mass spectral techniques. The mechanism of binding of these probes with calf thymus DNA (CT-DNA) has been delineated through UV-Vis, fluorescence, and circular dichroism (CD) spectral techniques along with thermodynamic and molecular docking studies. Small hypochromicity in absorption maximum of the probes without any shift in wavelength of absorption suggests groove binding mode of interaction of these probes with CT-DNA, confirmed by CD and 1H NMR spectral data competitive binding assay with ethidium bromide (EB). CT-DNA quenches the fluorescence of these probes via a static quenching mechanism. In the case of R1 and R4, the observed ΔHo < 0 and ΔSo > 0suggest that these probes interact with CT-DNA through H-bonding and hydrophobic interactions, while in the interaction of R2 and R3, van der Walls and H-boding forces are found to be dominant (ΔHo < 0 and ΔSo < 0). Results of molecular docking investigations corroborate well with that of spectral studies, and these probes bind in the minor groove of DNA. These probes are found to be effective fluorescent staining agents for DNA in agarose gel in gel electrophoresis experiment with sensitivity comparable to that of EB, and DNA amounts as low as 37.5 ng are visually detectable in the gel.

2-Aminophenols based Schiff bases as fluorescent probes for selective detection of cyanide and aluminium ions - Effect of substituents.

Three Schiff base based probes are designed and synthesized by facile condensation of a commercially available fluorophore 2,6-diformyl-4-tert-butylphenol with 4-nitro-2-aminophenol (KP1), 2-aminophenol (KP2) and 4-tert-butyl-2-aminophenol (KP3) and are characterized using various spectral techniques. The probes exhibit high selectivity and sensitivity CN- and Al(III) ions with striking fluorescent signaling responses in H2O-DMSO (1:1, v/v) medium. The mechanism of the probes' detection of CN involves deprotonation of the phenolic OH group(s) followed by nucleophilic addition of CN- onto imine C-atom. The 1H NMR chemical shifts of the OH protons of 2-aminophenol moiety exhibits a linear correlation with the Hammett's substituent constants (σp), yielding a positive reaction constant (ρ). In KP1, the electron-withdrawing nitro substituent polarizes the imine bond to a larger extent than in KP2, resulting in easier addition of CN- to imine C-atom. The electron releasing tert-Bu substituent in KP3 produces the opposite effect leading to a sluggish addition reaction. The separately populated HOMO and LUMO in KP1 and a relatively lower HOMO-LUMO energy gap indicate substantial intramolecular charge transfer (ICT) character, leading to weak fluorescence emission. The large reduction in HOMO-LUMO energy gap, in KP1, upon addition of cyanide is responsible for the greater enhancement in fluorescence with blue shift upon addition of CN-. Formation of tetrahedral Probe-Al(III) complex prevents the isomerization of imine bond, leading to enhancement in fluorescence and contribution from chelation enhanced fluorescence. As these probes show very low limits of detection of these ions, their practical utility has also been demonstrated.